1. Field of the Invention
The present invention relates to compounds for binding bio-affecting substances, such as therapeutic and diagnostic agents, to the surface membrane of viable bio-particles, including eukaryotic prokaryotic cells and viruses, without producing appreciable detrimental effects on morphology or physiological function of the bio-particle to which the compounds are bound. The invention also relates to compositions and methods enabling binding of such compounds to bio-particles and to the use of the resultant particles for producing a site-specific predetermined effect, e.g., a diagnostic or therapeutic effect, in vivo.
2. Background Information
Numerous compounds and compositions are known which are capable of binding bio-affecting substances to carrier cells for various diagnostic applications. Fluorescent labeling techniques using a wide range of fluorochromes have been reported, such as cyanine derivatives, U.S. Pat. No. 4,343,782, fluorescein isothiocyanate, Butcher et al., J. Immunol. Methods, 37, 109-21 (1980) and Butcher et al., J. Immunol. Methods, 37, 97-108 (1980) and fluoroscein or rhodamine having a single, relatively long aliphatic hydrocarbon substituent, Wanda et al., J. Histochem. Cytochem., 30, 1297-1300 (1982). There has also been reported a non-reproducible labeling technique using 3,3'-di-n-octodecyloxacarbocyanine for retrograde labeling of neurons Honig et al., J. Cell Biology, 103, 171-87 (1986). These prior art cell labelling compounds have been found to be: 1) unstable in the plasma membrane for long periods of time, and/or 2) cytotoxic or otherwise detrimental to cell function or morphology, and/or 3) not capable of providing reproducible results.
Diagnostic techniques utilizing chelate-metal ion complexes for radiographic and nuclear magnetic resonance imaging are fairly well developed. The use of complexes of Gallium-67, Indium-111, and Technetium-99 m in radio-imaging techniques have been widely reported. See, for example, Holman, ed., Radionuclide Imaging of the Brain (New York: Churchill Livingstone, 1985). The use of paramagnetic complexes in NMR imaging has been extensively investigated and compositions including such complexes have been proposed for administration as image enhancers. See, Australian Patent application 86 330/82 of Greis et al., filed Jul. 22, 1982. Insofar as is known, however, such imaging techniques do not result in membrane binding but rather the imaging complex is taken up into the cell and binds to the reticulum of the cell. In these applications, the chelating agent, e.g., oxine, tropolone or merc, have a toxic effect on many cells, such as lymphocytes, and are not optimal for imaging. Furthermore, since the detectable metal ion is taken into the cell, isotopic decay can more easily result in radiation damage to the cell's genetic material.
European Patent application Serial No. 86103694.5, published Oct. 22, 1986, describes a method for the selective irradiation of biological materials, particularly tissues, cells or cell components, in which the Mossbauer absorption frequency of a component of the material to be irradiated is determined and the material is then irradiated with gamma radiation of the corresponding Mossbauer absorption frequency with internal conversion and emission of gamma radiation and/or Auger electrons. The method is disclosed as being useful for selective radiation therapy providing selective tissue damage or necrosis, e.g., in cancer therapy, and for differentiating between diseased and healthy tissues. In practicing this method, Mossbauer isotopes are administered parenterally but are not selectively delivered to the tumor cells. It is believed that the selectivity in tumor cell kill will be achieved either through differences in absorption wavelengths of Mossbauer atoms within tumor cells and normal cells or by specifically focusing the absorbed radiation at the tumor itself.
One of the major goals of any pharmaceutical development is to provide drug therapy which is totally specific for a target cell type or disease site. In some cases, the compounds used are antagonists or agonists where specific receptors are found on cells to which therapy is directed and lower levels or affinities on other cell types. In other cases, the drug is specifically taken up by cells at the disease site or metabolized at the disease site in a way which is different from the matabolism at non-disease sites.
In treating cancer, many drug therapies proceed on the assumption that cancer cells are growing and metabolizing at a rate which is greater than most non-tumor cells. This assumption generally is flawed by the fact that hair follicles, bone marrow cells and gastro-intestinal cells grow at even faster rates and are often quite affected by these therapies. But even in cancer therapies, the goal is to try to deliver the therapy only to the tumor cells.
One recent attempt at this goal is to use monoclonal antibodies, as described in published European Patent application No. 83400461.6. With this therapy, the monoclonal antibody has "specific" binding affinity for the tumor cells. Radio-therapy or chemotherapeutic molecules are bound (covalent or ionic) to the monoclonal and injected intravenously into the patient. The monoclonal then migrates to the tumor site where it binds to the tumor cells with sufficiently high affinity to allow for the accumulation of radiation or chemical damage to be accrued by the tumor cells. This "therapy" is frought with a number of problems, not the least of which is the large amount of protein which must be injected for each treatment, making this therapy quite expensive. Additionally, the monoclonals injected are foreign protein and often results in the generation of antibodies by the patient against the therapeutic molecule. Furthermore, specificity of binding is often a problem and non specific toxicity is the result. Antigen shedding or modulation is equally problematic for this methodology, as well as limited capability to enter poorly vascularized tumors. In general, a methodology which could deliver a desired therapeutic effect specifically to tumor sites, is a desirable attribute for a pharmaceutical substance.
In copending U.S. patent application Ser. No. 925,192, filed Oct. 31, 1986 now U.S. Pat. No. 4,783,401, methods are disclosed for reproducibly labeling viable cells with symmetrical cyanine dyes that do not significantly affect cell viability. Applications for such labeled cells include using labeled red blood cells to distinguish post-transfusional bleeding from immunologic reaction and using dilution to measure growth rate of cultured cells.
In copending U.S. patent application Ser. No. 925,445, filed Oct. 31, 1986 now U.S. Pat. No. 4,762,701, methods are disclosed for tracking cells in vivo and for determining in vivo cell lifetimes. In performing such methods, cells are labeled with cyanine dyes and detection is by measuring fluorescence, absorbance, or by detecting nuclear magnetic reasonance probes included in the cyanine dyes. The methods are useful, for example, to measure red blood cell and platelet lifetimes, to track cells to determine sites of primary or metastatic tumors, or sites of occult infection, and to determine rates at which cells pass through vessels for assessing blood vessel patency and platelet aggregation.
In copending U.S. patent application Ser. No. 925,429, filed Oct. 31, 1986 now U.S. Pat. No. 4,859,584, methods are disclosed for determining growth rate of cells growing in vivo and in vitro. In carrying out such methods, cells are labeled with cyanine dyes and changes in plasma membrane cyanine dye levels are used to determine growth rate. The resulting cell growth rate determinations are utilized to monitor transplanted bone marrow cell engraftment and post-surgical corneal epithelial cell growth. Such methods also are useful for determining tumor cell sensitivity to cancer therapeutic agents, yeast sensitivity to antifungal agents, bacteria sensitivity to antifungal agents, and bacteria sensitivity to antibacterial agents.